The goal of this interdisciplinary program is to elucidate the mechanism of presynaptic membrane fusion from a mostly structural-biophysical perspective. Fusions of intracellular membranes in the secretory pathway are mediated by supramolecular machines that are assembled on demand and disassembled when the task is completed. At the core of presynaptic (and other intracellular) membrane fusion is the assembly of the SNARE core complex. The most prevalent model in the field is that the assembly of the SNARE core complex drives the membrane merger by a mechanism that is still poorly understood. The assembly and disassembly of SNAREs is believed to be regulated and catalyzed by numerous accessory proteins including SM-proteins, NSF, and alpha-SNAP. Although structures of soluble domains of many of these proteins and even of some complexes between them are known and have enormously contributed to our current understanding of their possible roles in membrane fusion, their precise sites of action at different steps of membrane docking and fusion have not yet been determined. Likewise, the site of action of the calcium sensor synaptotagmin in synaptic fusion is still poorly understood at the molecular level. Therefore, the main focus of this program is to shift attention to studying spatial relationships and interactions of these membrane proteins in the context of membranes. The three major program components will address how these fusion machines assemble/disassemble on membranes and work on membranes to eventually convert them to form productive fusion pores. To accomplish this goal, new biophysical techniques will be developed and used.